Molecular Dissection of the Essential Features of the Origin of Replication of the Second Vibrio Cholerae Chromosome

Abstract

Vibrionaceae family members are interesting models for studying DNA replication initiation as they contain two circular chromosomes. Chromosome II (chrII) replication is governed by two evolutionarily unique, yet highly conserved elements: the origin DNA sequence, oriCII, and the initiator protein, RctB. The minimum functional region of oriCII, oriCII-min, contains multiple elements that are bound by RctB in vitro, but little is known about the specific requirements for individual elements during oriCII initiation. We utilized undirected and site-specific mutagenesis to investigate the functionality of oriCII-min mutants, and assessed binding to various mutants by RctB by electrophoretic mobility shift assays. Our analyses showed that deletions, point mutations, and changes in RctB target site spacing or methylation all impaired oriCII-min-based replication. RctB displayed reduced affinity for most low efficacy origins tested, although its characteristic cooperative binding was generally maintained. Mutations that removed or altered the relative positions of origin components other than RctB binding sites (e.g., AT-rich region, DnaA target site) also abolished replicative capacity. A comprehensive mutagenesis and deep-sequencing-based screen (OriSeq) allowed identification of a previously uncharacterized methylated domain in oriCII that is required for origin function. We also examined RctB-oriCII interactions with DNaseI footprinting, pelleting, and electron microscopy. Along with the binding studies, these data suggest that RctB is able to generate large, oligomeric structures on DNA, which are sequence independent at high proteins concentrations. Together, our results reveal the remarkable evolutionary honing of oriCII and provide new insight into the complex interplay between RctB and oriCII.